Process for the preparation of polyprenyl ketone derivatives

ABSTRACT

Longer carbon chain polyprenyl ketone derivatives are prepared by  cultivag Nocardia strain FERM-P No. 1609 in the presence of shorter carbon chain polyprenyl ketones.

This is a division of application Ser. No. 312,068, filed Oct. 16, 1981.

This invention relates to novel polyprenyl ketone derivatives having thegeneral formula: ##STR1## in which n is an integer of 1-5, and Rrepresents a hydroxymethyl, formyl or carboxyl group, and a process forthe preparation of the same.

Polyprenyl-type compounds such as polyprenyl alcohols and their esters,polyprenyl carboxylic acids and their esters, and polyprenyl ketones areknown to have the antiulcer activity and the hypotensive activity, asdisclosed in Japanese Patent Provisional Publications No. 52-144614, No.53-145922, No. 54-5043, No. 54-67037, and No. 54-76513. Further,polyprenyl alcohols are known to be employable as starting materials forthe preparations of pharmaceutically active compounds such as CoenzymeQ₁₀.

The compound of the present invention is of value as an intermediatecompound for preparing the above-mentioned polyprenyl-type compounds,and also is of value per se as an anti-ulcer agent.

The compound of the invention can be converted to the polyprenyl-typecompounds, for instance, via a sulfone derivative as disclosed inJapanese Patent Provisional Publications No. 53-103444 and No.53-103445. The compound of the invention can be employed as anintermediate compound for the synthesis process, in the original formwhere R is the hydroxymethyl group, or in the form of the hydroxymethylgroup upon reduction where R is the formyl or carboxyl group.

The reactions can be illustrated, for instance, by the followingreaction equations.

(1) Polyprenyl ketone ##STR2##

In the above reaction equations, n has the same meaning as above, m isan integer, and Y represents an aryl or alkyl group.

(2) Polyprenyl carboxylic acid and its derivative ##STR3##

In the above reaction equations, n amd m have the same meanings asabove, and R₁ represents a lower alkyl group.

(3) Polyprenyl alcohol ##STR4##

In the above reaction equations, n, m and R₁ have the same meanings asabove.

As shown in the above (1)-(3) sections, the employment of the compoundof the invention in the preparation of polyprenyl-type compounds canprolong the carbon chain to an optionally selected level in terms of thecarbon number.

As stated hereinbefore, various compounds having a functional group atthe terminal of their isoprenoid chain are known. However, the selectiveintroduction of a functional group into another terminal of the abovecompound is difficult from the view point of the chemical synthesisprocess. This difficulty increases where the isoprenyl chain isrelatively long. Accordingly, the present invention provides a processfor the preparation of the subject compound involving microbiologicaloxidation. More in detail, the compound of the invention can be obtainedby microbiological oxidation of polyprenyl ketone by the use of amicroorganism belonging to the genus Nocardia.

The genus Nocardia named BPM 1613 was deposited with the FermentationResearch Institute, the Agency of Industrial Science and Technology,located at 1-1-3, Higashi, Tsukuba-Yatabe-machi, Ibaraki-prefecture,Japan on Sept. 18, 1972 and has been added to its permanent collectionof microorganisms as FERM-P No. 1609. The same genus Nocardia as abovewas deposited also with the Institute for Fermentation, Osaka, locatedat 2-17-15, Juso-Honmachi, Yodogawa-ku, Osaka, Japan on Jan. 9, 1981 andhas been added to its permanent collection of microorganisms as IFO14101. This strain can be employed in the invention and has particularsas given below. The color is expressed according to the "Color Standard"published by Nippon Shikisai Kenkyusho (Japan Color Research Center),Japan.

A. Form of Cells

The present strain shows characteristic orange to pink color in almostall culture media, as seen from the cultural characteristics givenbelow. A young vegetative cell grows in a mycelial form, and thebranching is rarely observed. In an aged cultivated system, the hypha isdivided to form a bacillus (0.4-0.6×1.8-2.4 μ). Gram positive. Noflagellum. Negative on the acid-fast stain according to theZiehl-Nielsen method. Aerial mycelium is not observed.

B. Cultural Characteristics on Various Media

(1) Sucross - Nitrate Agar Medium (30° C.): poor growth; pink coloredcolony; no diffusive pigment

(2) Glucose - Asparagine Agar Medium (30° C.): no growth

(3) Glycerol - Asparagine Agar Medium (30° C.): poor growth; pinkcolored colony; no diffusive pigment

(4) Starch Agar Medium (30° C.): no growth

(5) Tyrosine Agar Medium (30° C.): poor growth; grayish white coloredcolony; no diffusive pigment

(6) Nutrient Agar Medium (30° C.): moderate growth; orange coloredcolony; no diffusive pigment

(7) Yeast - Malt Agar Medium (30° C.): rich growth; orange coloredcolony; no diffusive pigment

(8) Oatmeal Agar Medium (30° C.): moderate growth; orange coloredcolony; no diffusive pigment

(9) Calcium Maleate Agar Medium (27° C.): moderate growth; pink coloredcolony

(10) Egg-albumin Medium (slant, 27° C.): poor growth; white colony

(11) Potato Section Medium (27° C.): moderate growth; pale orangecolored colony

(12) Carrot Section Medium (27° C.): moderate growth; pale pink coloredcolony

C. Physiological characteristics

(1) Growth Temperature Range (on Nutrient Agar Medium, slant): 20°-42°C.

(2) Liquefaction of Gelatin: megative

(3) Hydrolysis of Starch: negative

(4) Coagulation of Defatted Milk, Peptonization: negative

(5) Litmus Milk: no change

(6) Production of Melanine-like Pigment: negative

(7) Reduction of Nitrate: positive

(8) No gas or acid production from L-arabinose, D-xylose, D-glucose,D-fructose, sucrose, D-mannitol, glycerol, lactose, D-galactose,D-mannose, maltose, trehalose and starch

(9) Catalase Test: negative

(10) Production of Indole: negative

(11) Production of Hydrogen Sulfide: negative

C. Assimilability for Various Carbon Sources

(Pridham-Gottlieb Agar Medium, 30° C., 7 days) L-arabinose (+), D-xylose(+), D-glucose (++),

D-fructose (++), sucrose (++), inositol (+),

L-rhamnose (-), raffinose (+), D-mannitol (+) (In the above, (++) meansmoderate growth,

(+) means poor growth, and (-) means no growth)

The above-identified strain, having been cultivated on theGlycerol-Kelner-Morton Medium in accordance with the method described byArai, et al., in Journal of General Applied Microbiology, 9, 119 (1963):The Actinomycetales, The Jena International Symposium on Taxonomy, 273(1968), gives absorption bands characteristic of the genus Nocardia onthe IR spectrum, that is, I: C & E types, II: C type, III: C type, andIV: D type.

Upon studying the above-described characteristics of the strain withreference to Bergey's Manual of Determinative Bacteriology, Seventhedition, and Waksman's The Actinomycetes, Volume 2, it has been decidedthat the strain belongs to the genus Nocardia.

A process for the preparation of the compound of the invention isdescribed below.

The compound of the invention having the general formula: ##STR5## inwhich n is an integer of 1-5, and R represents a hydroxymethyl, formylor carboxyl group can be obtained by cultivating a microorganismbelonging to the genus Nocardia and showing the oxidizing activity on acompound having the general formula: ##STR6## in which n has the samemeaning as above in a culture medium containing a compound of thegeneral formula (II), and recovering the oxidized product from thecultured medium.

The cultivation procedure is described more concretely hereinbelow.

The components of the culture medium can be optionally chosen from thoseemployed conventionally, except for the necessary inclusion of thecompound of the general formula (II) as the carbon source. Examples ofthe nitrogen sources include nitrates such as potassium nitrate, sodiumnitrate and ammonium nitrate; ammonium salts such as ammonium chloride,ammonium sulfate and ammonium phosphate; ammonia; and urea. Ifnecessary, inorganic salts such as potassium phosphate, sodiumphosphate, magnesium sulfate, ferrous sulfate and manganese sulfate; andorganic nutrient sources such as vitamins and amino acids; and yeastextract, corn steep liquor and malt extract which contain the vitaminsand amino acids can be incorporated, as well.

The medium is preferably adjusted to have the pH value in the range of6-8. The cultivation is generally carried out under aerobic conditionssuch as under aeration and agitation at 20°-40° C. for 2-5 days.

After the cultivation is complete, the cultivation product is extractedwith an organic solvent to recover the compound of the invention.Examples of the solvents for extraction include ethyl ether, benzene andchloroform. The product can be subjected to the silica gel columnchromatography to separate and purify the compound of the invention.

The unreacted starting material can be recovered by the above-describedextracting procedure and column chromatography, and then can becirculated as the starting material for the following run.

The microbiological oxidation of the process of the invention gives amixture of different products having hydroxymethyl, formyl and carboxylgroups, independently, as the terminal group (R of the general formula(I)), depending upon the extent of the oxidation. The constitution ofthe product, accordingly, can be varied depending upon the kind of theculture medium, the cultivation period, and so forth.

The present invention is further described more in detail by thefollowing examples.

EXAMPLE 1 ##STR7##

The strain (BPM 1613, FERM-P No. 1609) belonging to the genus Nocardiawas precultivated under shaking at 30° C. for 4 days in 50 ml of amedium comprising 2% of n-paraffin, 0.5% of NaNO₃, 0.15% of KH₂ PO₄,0.15% of Na₂ HPO₄, 0.05% of MgSO₄.7H₂ O, 0.001% of FeSO₄.7H₂ O, 0.001%of CaCl₂.2H₂ O, and 0.02% of yeast extract and having the pH value of7.2

Then, the so obtained precultural broth was inoculated, in the volumeratio of 8%, into a jar fermenter (for fermentation of 1 liter medium):a medium having the same composition as defined above except forsubstituting the n-paraffin (2%) by the compound of the general formula(II) (1%). The cultivation was carried out under aerationagitationconditions at 30° C. for 3 days. After the cultivation was complete, thecultivation product was extracted with ethyl ether. The thin layerchromatograpny of the extract indicated productions of the compoundshaving hydroxymethyl, formyl and carboxyl groups, respectively, as theterminal R group.

The solvent was then removed from the ethyl ether extract byevaporation, and the residue was purified by the silica gel columnchromatography to isolate the main products. In this procedure, hexaneand ethyl ether were employed as the eluting solvents.

The results obtained in the above process are set forth in Table 1. Itwas found that by-products were hardly produced and almost all thestarting material that had been actually reacted was converted to thedesired product. Almost all unconverted starting material could berecovered with the silica gel column chromatography as mentioned above.

In the example, the following compounds were produced.

10-keto-2,6-dimethyl undeca-2,6-dien-1-carboxylic acid

14-keto-2,6,10-trimethyl pentadeca-2,6,10-trien-1-carboxylic acid

18-keto-2,6,10,14-tetramethyl nonadeca-2,6,10,14-tetraen-1-carboxylicacid

22-keto-2,6,10,14,18-pentamethyltricosa-2,6,10,14,18-pentaen-1-carboxylic acid

26-keto-2,6,10,14,18,22-hexamethylheptacosa-2,6,10,14,18,22-hexaen-1-carboxylic acid

10-keto-2,6-dimethyl-undeca-2,6-dien-1-ol

14-keto-2,6,10-trimethyl-pentadeca-2,6,10-trien-1-ol

18-keto-2,6,10,14-tetramethyl nonadeca-2,6,10,14-tetraen-1- ol

22-keto-2,6,10,14,18-pentamethyl tricosa-2,6,10,14,18-pentaen-1-ol

26-keto-:2,6,10,14,18,22-hexamethylheptacosa-2,6,10,14,18,22-hexaen-1-ol

18-keto-2,6,10,14-tetramethyl nonadeca-2,6,10,14-tetraen-1-al

22-keto-2,6,10,14,18-pentamethyl tricosa-2,6,10,14,18-pentaen-1-al

                                      TABLE 1                                     __________________________________________________________________________     ##STR8##                                                                             State                                                                             Mass                                                              Product (I)                                                                           Yield                                                                             Spectrum                                                          n R     (%) (M.sup.+)                                                                          NMR Spectrum (δ, CDCl.sub.3)                           __________________________________________________________________________    1 HOOC  Oil 224  1.69 (3H, s), 1.84 (3H, s), 1.93˜2.55 (8H, br),                         2.10 (3H, s),                                                        10.1     5.05 (1H, t, J = 6Hz), 6.87 (1H, t, J = 7Hz), 11.5 (1H,                       br)                                                          1 HOH.sub.2 C                                                                         Oil 210  1.62 (3H, s), 1.66 (3H, s), 1.95˜2.50 (9H, br),                         2.09 (3H, s),                                                         2.8     3.94 (2H, s), 5.04 (1H, t, J = 6Hz), 5.30 (1H, t, J =                         7Hz)                                                         2 HOOC  Oil 292  1.60 (6H, s), 183 (3H, s), 1.95˜2.50 (12H, br),                         2.10 (3H, s),                                                        12.6     4.95˜5.15 (2H, br), 6.88 (1H, t, J = 6Hz),                              11.0˜12.0 (1H, br)                                     2 HOH.sub.2 C                                                                         Oil 278  1.60 (6H, s), 1.65 (3H, s), 1.95˜2.50 (13H, br),                        2.10 (3H, s),                                                         3.2     3.95 (2H, s), 4.95˜5.15 (2H, br), 5.30 (1H, t, J =                      6Hz)                                                         3 HOOC  Oil 360  1.60 (9H, s), 1.83 (3H, s), 1.95˜2.50 (16H, br),                        2.10 (3H, s),                                                        16.2     5.00˜5.20 (3H, br), 6.87 (1H, t, J = 7Hz),                              9.50˜10.0 (1H, br)                                     3 HOH.sub.2 C                                                                         Oil 346  1.60 (9H, s), 1.65 (3H, s), 1.95˜2.50 (17H, br),                        2.09 (3H, s),                                                        32.1     3.95 (2H, s), 5.00˜5.20 (3H, br), 5.30 (1H, t, J =                      6Hz)                                                         3 OHC   Oil 344  1.60 (9H, s), 1.75 (3H, s), 1.95˜2.50 (16H, br),                        2.10 (3H, s),                                                         1.1     5.10 (3H, br), 6.46 (1H, t, J = 6Hz), 9.50 (1H, s)           4 HOOC  Wax 428  1.60 (12H, s), 1.81 (3H, s), 1.95˜2.50 (20H, br),                       2.10 (3H, s),                                                        19.7     4.90˜5.15 (4H, br), 6.90 (1H, t, J = 7Hz),                              11.5-12.0 (1H, br)                                           4 HOH.sub.2 C                                                                         Wax 414  1.60 (12H, s), 1.65 (3H, s), 1.95˜2.50 (21H, br),                       2.10 (3H, s),                                                        14.6     3.95 (2H, s), 4.90˜5.15 (4H, br), 5.30 (1H, t, J =                      7Hz)                                                         4 OHC   Wax 412  1.60 (12H, s), 1.76 (3H, s), 1.95˜2.50 (20H, br),                       2.10 (3H, s),                                                         1.6     5.10 (4H, br), 6.47 (1H, t, J = 6Hz), 9.48 (1H, s)           5 HOOC  Wax 496  1.60 (15H, s), 1.81 (3H, s), 1.95˜2.50 (24H, br),                       2.08 (3H, s),                                                        20.2     4.90˜5.15 (5H, br), 6.90 (1H, t, J = 6Hz),                              11.0˜12.0 (1H, br)                                     5 HOH.sub.2 C                                                                         Wax 482  1.60 (15H, s), 1.65 (15H, s), 1.95˜2.50 (25H, br),                      2.09 (3H, s),                                                        18.3     3.95 (2H, s), 4.90˜5.15 (5H, br), 5.30 (1H, t, J =                      6Hz)                                                         __________________________________________________________________________

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A process for thepreparation of a polyprenyl ketone derivative having the generalformula: ##STR9## in which n is an integer of 1-5, and R represents ahydroxymethyl, formyl or carboxyl group, which comprises:cultivating amicroorganism belonging to the genus Nocardia identified as BPM 1613,FERM-P No. 1609, which shows an oxidizing activity on a compound havingthe general formula: ##STR10## in which n is an integer of 1-5, in aculture medium containing a compound of the general formula (II); andrecovering the oxidized product from the cultured medium.
 2. A processas claimed in claim 1 in which the culture medium has a pH of from 6 to8 and the cultivating step is performed under aerobic conditions, at 20°to 40° C., for from 2 to 5 days.